Map Kinase Kinase Kinase Kinase Cascades

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Mitogen activated protein kinase (MAPK) cascades

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three kinases that sequentially activate each other
(name reflects level in cascade)

spatio-temporal control mechanisms of MAPK explain how so many cellular processes can be regulated

MAPKs are CMGC Ser/Thr kinases that require double phosphorylation on Thr and Tyr for full activation (Lecture ST1).

This is done by the MAP2K that are double-specificity kinases.

They themselves often require double phosphorylation on Ser and Thr by MAP3Ks.

MAP3Ks are activated by a variety of input signals that are controlled by various pathways that include receptors, small GTPases, G-proteins, etc.

All MAP3K invariably require activation by an upstream kinase that sometimes is called MAP4K. These kinases however belong to various different classes.

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how do MAPKs regulate gene expression

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MAP kinases are essentially “naked” kinase domains with short flanking C and N termini. Once activated by phosphorylation, they detach from the MAP2Ks, dimerize and become fully active signal transducers


-active MAPK dimers can travel to the nucleus and affect gene expression levels by phophorylating transcription factors (eg TCF/ Elk)
- multiple targets in PM, cytoplasm & nucleus &other organelles
=> may or may not affect gene expression
(ribosomal protein S6 kinases can directly phosph. SRF trans. factors)

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Yeast pheromone pathway

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pheromone receptors=> Ste11-Ste7-Fus3 kinases relay signal to the nucleus via Ste12 activation

Pheromone receptor >>> Cdc42 (small GTPase) + Gβγ subunit => Ste20 kinase=> Ste11-Ste7-Fus3 kinase cascade=> Ste12 (transcription factor)=> mating genes activated

**Gβγ + PIP2 lipid => Ste5 (scaffold that recruits Ste11-Ste7-Fus3)

=> several factors need to be activated together for activation of this cascade (G-proteins, PIP lipids, small Rho GTPases

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Role of scaffolds in MAPK signalling

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bring together kinases and their activators
=> drastically change kinetics in several ways

* MAPK Kss1 is activated by Ste11-Ste7, in the absence of Ste5 scaffold, causing filamentous growth

WHY?
W. Lim et al demonstrated that Ste5 is necessary for the activation of Fus3 by unlocking its activation loop, allowing for phophorylation by Ste7

Ste11 is constitutively bound to Ste50 adaptor/scaffold?, which is required for Ste11 function
Ste50 binds Cdc42, recruiting Ste11 to the membrane

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Morphogenesis in Yeast is controlled by two MAPK kinases

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Fus3 and Kss1 collaborate and compete in controlling yeast morphogenetic programs
* are evolutionarily connected

Ste5 present:
Ste11 => Fus3

Ste5 absent:
Ste11 => Kss1

Both Fus3 and Kss1 are activated as a pheromone response

Only Kss1 is activated in response to nutrient starvation ==> filamentous growth

Tec1+Ste12 => filamentous growth genes

Ste12 alone=> mating genes

Fus3 degrades Tec1 => Ste12 alone => mating genes
*prevents cross activation

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Cellular stress responses in yeast controlled by two MAPK cascades

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various stresses (high/low osmolarity, temperature, oxidants etc) => trigger two MAPK cascades:
HOG (high osmolarity glycerol)
CWI (cell wall integrity)

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HOG pathway

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high osmolarity glyerol

activated by two branches that converge on Ste11
and Ssk2-Ssk22 MAP3Ks

both activate MAP2K Pbs2, which also acts as a scaffold for the same pathway

Hog1 MAPK is the effector kinase of the HOG pathway
=> regulates multiple stress response pathways

CWI uses totally distinct components
activated by MAP4K Pkc1, downstream of small GTPase Rho1
- pathway activated by various stress factors, at various stages along the pathway

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Smk1

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Firth MAPK cascade, activated by nitrogen starvation (only during meiosis) => regulates sporulation

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Mammalian MAPK cascades

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-generally much more complex than yeast

- preserve general three-tier organisation

ERK
=> cell cycle, growth and morohogenesis (analogous to Fuse-Kss1)

p38 & JNK
=> sense various stresses, analogous to Hog1 and Mpk1 MAPKs
=> pleiotropic roles, either pro-survival or pro-apoptotic

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JNK

ERK

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c-Jun N-terminal kinase

extracellular regulated kinases

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ERK

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activated by various signalling molecules
best known pathway: downstream of EGF receptors

EGF=> Grb2 and SOS signalling complex (Ras GEF) => Ras => recruit + activate MAP3K Raf

*both ERK1,2 and Mek can dimerize
*PAK kinases play the MAP4K role

scaffolding proteins (KSR, paxilin p14) give additional complexity to ERK pathways
HOW?
-> activate ERK at different intracellular locations=> result in different cellular responses

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How does the structure of MAPK cascades generate complex behaviours

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dose response curve of signal systems

LINEAR are the systems that behave in the Michaelis-Menten law.

ULTRASENSITIVE: described by the hill law (n>1)
=> enables noise supression
=> steep rise upon reaching threshold level of input.

J. Ferrell found that MAPK cascade activity may generate ultrasensitive responses with Hill coefficients of ~5

*many molecular mechanisms can explain same experimental data, not certain if it is ultrasensitive indeed

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Feedback loops shape MAPK cascade dynamics

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perfect adaptation:
sharp drop to prestimulus level after activation (steep rise)
steep rise followed by sharp drop

how?

one or more +ive feedback loops amplify response when certain threshold has been exceeded by
1) removal of pre-coupled phosphatases (signal supressing)
2) activation of upstream components

negative feedback => disassembly of the cascade in response to phosphrylation of the phophatases

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synthetic biology MAPK cascades

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Wendell Lim et al :

Ste5 yeast protein expressed with sticky bit